Dentinogenesis is regulated by a single layer of highly differentiated post-mitotic odontoblasts originating from the dental papilla. Despite significant progress in the discovery of key molecules and signaling pathways regulating tooth initiation and patterning, the mechanisms leading to dentinogenesis are relatively unknown. This has been due, in part, to difficulties in obtaining homogenous populations of progenitor cells for molecular analysis and the lack of suitable molecular markers for identifying the intermediate stages of odontoblast differentiation. The overall goals of this proposal are to use promoter-GFP reporter transgenic mice as a novel experimental model to unravel the underlying molecular mechanisms regulating the linear stepwise progression of pulp progenitor cells into odontoblasts. This proposal will use the activation of Col1a1-GFP transgenes to identify and isolate cells at early stages of differentiation by FACS sorting for further cellular and molecular analyses including an examination of their differentiation potential and gene expression profile. 3 specific aims are proposed. Aim #1 will test the hypothesis that stage-specific activation of Col1a1-GFP transgenes can serve as markers to distinguish and identify sub-populations of cells from dental pulp for further lineage analysis in vitro. Aim #2 will test the hypothesis that insulin-like growth factor (IGF-I) stimulates odontoblast differentiation. In Aim 2, Col1a1-GFP transgenes will be used to examine the underlying mechanisms by which IGF-I stimulates odontoblast differentiation. In this aim the effects of the altered levels of IGF-I on proliferation, apoptosis, and differentiation of pulp cells derived from transgenic mice with bone/odontoblast targeted over-expression of IGF-I (pOBCo!3.6-IGF), and transgenic mice with IGF-I haploinsufficiency (Igf1, HET) will also be examined. Aim #3 will test the hypothesis that Col1a1-GFP transgenes can be used as a marker to assess the dentin forming potential of adherent pulp fibroblasts in an in vivo transplantation assay at an ectopic site. It can be envisioned that results obtained from these experiments, will provide fundamental insights in the development of stem cell-based treatments for the repair and regeneration of dentin and eventually whole tooth that has been compromised by congenital disorders, diseases, and injuries. [unreadable] [unreadable] [unreadable]